HUT67914A - Polymer-bound paclitaxel derivatives process for producing them and pharmaceutical compositions containing them - Google Patents

Abstract

A polymer conjugate consisting essentially of: from 90 to 99.9 mol % of units represented by the formula <IMAGE> from 0.1 to 5 mol % of units represented by the formula <IMAGE> wherein one of R1 and R2 is a copolymer residue of the formula <IMAGE> and the other one is hydrogen atom; from 0 to 9.9 mol % of units represented by the formula <IMAGE> wherein R is a phenyl or t-butoxy group, R3 is H or an acetyl group, A and A1 which may be the same or different, represent a chemical single bond, an amino acid residue or peptide spacer selected from beta Ala, Gly, Phe-Gly, Phe-Phe-, Leu-Gly, Val-Ala, Phe-Ala, Leu-Phe, Leu-Ala, Phe-Leu-Gly, Phe-Phe-Leu, Leu-Leu-Gly, Phe-Tyr-Ala, Phe-Gly-Phe, Phe-Phe-Gly, Phe-Leu-Gly-Phe, Gly-Phe-Leu-Gly-Phe,- beta Ala, Phe-Gly- beta Ala, Phe-Phe- beta Ala, Leu-Gly- beta Ala, Val-Ala- beta Ala, Phe-Ala- beta Ala, Leu-Phe- beta Ala, Leu-Gly- beta Ala, Phe-Leu-Gly- beta Ala, Phe-Phe-Leu beta Ala, Leu-Leu-Gly- beta Ala, Phe-Tyr-Ala- beta Ala, Phe-Gly-Phe- beta , Phe-Phe-Gly beta Ala, Phe-Leu-Gly-Phe- beta Ala or Gly-Phe-Leu-Gly-Phe- beta Ala. The compounds are endowed with antitumor activity and show improved water solubility and decreased toxicity in comparison with paclitaxel or its known analogs. A method for their preparation and pharmaceutical compositions containing them are also described.

Description

The present invention relates to polymeric paclitaxel and paclitaxel derivatives having antitumor activity, to processes for their preparation and to pharmaceutical compositions containing them as active ingredients.

Paclitaxel (also referred to as Taxol in many publications) is a member of the taxane family of diterpenes. The compound was isolated from the bark of Taxus brevifolia L. strain and characterized. Also known are paclitaxel analogs prepared from 10-desacetyl baccatin III from the needle of Taxus baccata L. by semi-synthetic means (See Wain et al., 1971, 93, 2325). These compounds have antitumor activity, but the compounds have very low water solubility and exhibit some toxic side effects when administered by injection or intravenous infusion using Cremophor EL® vehicle.

The present invention relates to polymeric conjugates comprising the following units:

90-99.9 mol% of the unit of formula (A);

CH ₂

CH ₃ -C-CO-NH-CH ₂ -CHOH-CH ₃ (A)

0.1 to 5 mol% of a unit of formula (B) in which

One of R1 and R2 is a copolymer of formula (C),

CH ₂

CH 3 -C-CONH-CH ₂ -CO-A- (C) while the other is hydrogen; and

0 to 9.9 mol% of the compound of formula D,

I

CH ₂

I

CH 3 -C-CONH-CH ₂ -CO-A] -NH-CH ₂ -CHOH-CH 3

I (D) where in the general formulas

R is phenyl or tert-butoxy,

R ₃ is hydrogen or acetyl,

A and Αχ, whether identical or different, represent a single bond, an amino acid residue, or one of the following peptide spacer groups (hydro moieties): 3Ala, Gly, Phe-Gly,

Phe-Phe, Leu-Gly, Val-Ala, Phe-Ala, Leu-Phe,

Leu-Ala, Phe-Leu-Gly, Phe-Phe-Leu,

Leu-Leu-Gly, Phe-Tyr-Ala, Phe-Gly-Phe,

Phe-Phe-Gly, Phe-Leu-Gly-Phe,

Gly-Phe-Leu-Gly-Phe, Gly-βΑΙβ, Phe-Gly-pAla,

Phe-Phe-3Ala, Leu-Gly-PAla, Val-Ala-0Ala,

Phe-Ala-3Ala, Leu-Phe-PAla, Leu-Gly-3Ala,

Phe-Leu-Gly-Ala,

Leu-Leu-Gly-Ala,

Phe-Gly-Phe ^ Ala,

Phe-Phe-Leu-Ala,

Phe-Tyr-Ala-Ala,

Phe-Phe-Gly-Ala,

You're Phe-Leu-Gly-Phe-pAla

Gly-Phe-Leu-Gly-Phe-Ala.

More particularly, the invention relates to polymeric conjugates of paclitaxel and paclitaxel derivatives which have increased water solubility and reduced toxicity.

Preferably, the mole% of the units containing paclitaxel or paclitaxel derivatives is in the range of 0.5 to 2, more preferably the polymer has a paclitaxel content of 2 to 10% by weight, and most preferably the compounds having 4 to 7% by weight containing paclitaxel. The wavy line in formula (B) is intended to indicate that the 7-position oxygen atom of the paclitaxel molecule may be present in either configuration, i.e., it may be either β (natural) or spatial.

According to a preferred solution

R is phenyl,

R3 is acetyl and

A is Phe-Leu-Gly or Phe-Leu-Gly-pAla.

All occurring amino acid residues have the natural L-configuration.

In a further preferred embodiment, the polymeric conjugate is 1- (methacryloylamino) -2-hydroxypropane, [methacryloylglycyl (phenylalanyl) leucylglycyl] -3-amino-2-hydroxypropane and a copolymer of 2 '- [methacryloyl-glycyl- (phenyl-alanyl) -leucyl-glycyl-Palanyl] -paclitaxel.

The weight% content after enzymatic hydrolysis was determined by high performance liquid chromatography (HPLC) as previously described in Cancer Treatment Rep., 71 (1), 53-59 (1987).

Enzymatic hydrolysis

Polymer-bound paclitaxel was added to 1 ml volumes of Murine (mouse / rat) or human plasma, and 100 μ mint samples were taken at predetermined times (24, 48, 72, 96 hours) until further analysis at -70 ° C. temperature.

Extraction procedure

Each sample was extracted by first adding 75 μΐ 0.5 M tetrabutylammonium phosphate solution (TBAP), 1250 μΐ acetonitrile and 150 μΐ 5 M sodium chloride solution, and then vigorously for 20 minutes at 4 ° C. shaken.

Subsequently, the samples were exposed for 10 minutes

After centrifugation at 000 x g, the supernatants were removed and evaporated using a high vacuum centrifuge. The samples thus obtained were taken up in a volume of 500 μΐ of methanol / water (75:25) and injected into a high performance liquid chromatography (HPLC) to determine the percentage of total paclitaxel.

High performance liquid chromatography system

column: Nova Pák Cpg (Waters), 3.9 x 300 mL Flow rate: 1.5 ml / min Detector: UV 231 nm injection 20 μΐ Mobile phase 57.5% water (adjusted to pH 2)

42.5 / acetonitrile ·· ···· ···· ···· • "• · 4 · ··" ··· "•" • · · · «* · · ·

The invention also relates to a process for the preparation of polymeric conjugates comprising the step of:

One of ₂ and A3 has the meaning of a chemical bond, while the other has the same meaning as A and

A, R and R3 are as defined above - in an amount of 90-99.9 mol% from a unit of formula (A)

I

CH ₂

CH ₃ -C-CO-NH-CH ₂ -CHOH-CH 3 (A) and from 10 to 0.1 mol% of a unit of formula (E)

I

CHP

I

CH ₃ -C-CO-NH-CH ₂ -CO-Αχ-Ο- (1,4-phenylene) -NO ₃ (E) - in the formula

R 4 is reacted with a standing activated polymer as defined above and the resulting polymer conjugate is then reacted with 2-hydroxypropylamine.

Preferably, the reaction of the compounds of formula II with the activated polymer is carried out in an anhydrous polar organic solvent such as dimethylsulfoxide or dimethylformamide, optionally in the presence of an organic or inorganic base such as an alkaline carbonate, dimethylaminopyridine or triethylamine. carried out. The reaction is usually complete within 1-24 hours.

The reaction is usually carried out at a temperature of 15-40 ° C, preferably at room temperature. The alkali carbonate may be, for example, an alkali metal carbonate or an alkaline earth metal carbonate.

Some of the starting materials of formula (II) are known compounds, such as paclitaxel or paclitaxel analogs, or may be prepared from known compounds.

For example, 7-epi derivatives may be prepared by refluxing paclitaxel or paclitaxel analogs in toluene in the presence of a base (sodium carbonate or diazabicycloundecene).

However, other compounds of formula (II) are new, especially those in which

A3 is a PAla group and those in which the di-, tri- or tetrapeptide hydromolecular moieties (spacers) defined above for A are as defined above. ).

These compounds are also within the scope of the invention.

Compounds of formula II wherein:

₂ is a non-chemical single bond, may be prepared by reacting paclitaxel or a paclitaxel analogue with a protected amino acid or protected peptide in the presence of a condensing agent and optionally additional catalyst, preferably at room temperature, and then deprotecting in a known manner.

The condensation may also be carried out with activated esters of the peptide or amino acid, such as para-nitrophenyl ester. Suitable condensing reagents include carbodiimides such as dicyclohexylcarbodiimide (DCC).

Suitable catalysts include, for example, 4- (dimethylamino) pyridine (DMAP), pyridine or triethylamine.

Various protecting groups may be used in the starting materials to protect the amino groups, or commercially available starting materials in which the amino acid or peptides are already protected. Suitable peptides or amino acids for use include t-BOC (tert-butoxycarbonyl), trityl, FMOC (fluorenylmethoxycarbonyl) or CBZ (benzyloxycarbonyl) protecting groups. They contain. Preferred are amino acids and peptides protected with tert-butoxycarbonyl, trityl or fluorenylmethoxycarbonyl.

Compounds of formula II wherein:

A3 is a non-chemical single bond, can be prepared by protecting or blocking the 2'-hydroxy group, followed by esterification of the hydroxy group at position 7 and removal of the protecting or blocking group for the 2'-hydroxy group.

More preferably, the compounds of formula (II) wherein

A3 is a Gly or PAla group, may be prepared by the reaction of paclitaxel with 2-3 equivalents of the N-protected amino acid to synthesize 2 ', 7-disubstituted paclitaxel, and then cleave the amino acid at position 2'. amino acid protecting group.

The reaction of paclitaxel with the protected amino acid is carried out in the presence of a condensing reagent and a catalyst as described above.

To cleave the 2 'amino acid, the pH of the 2', 7-di (amino acid) -paclitaxel solution is adjusted to 7-7.4, e.g. or with a small excess of sodium bicarbonate.

Removal of the protecting groups of the above amino acids is accomplished by methods well known in the art of liberating protected amino acids, such as by treatment with a slight acid such as acetic acid or reduction.

For example, paclitaxel is indicated

2-3 equivalents (tert-butoxycarbonyl, benzyloxycarbonyl or fluorenylmethoxycarbonyl) of the N-protected amino acid in methylene chloride, dicyclohexylcarbodiimide (DCC) and catalytic amount of 4- ( in the presence of dimethylamino) pyridine. In this way, the protected amino acid is introduced at positions 2 'and 7. The 27-bis (amino acid) derivative of paclitaxel was allowed to stand in a water / methanol solvent mixture in the presence of sodium bicarbonate for 2 to 5 hours to give the 7-substituted derivative of paclitaxel as a result of selective cleavage at the 2 'position. The protecting groups are then removed using a suitable deprotecting agent (e.g. acid, weak base) or by hydrogenolysis.

Activated polymers are water-soluble polymers prepared by copolymerization of p-nitrophenyl esters of ek'- (2-hydroxypropyl) methacrylamide and N-methacryloyl oligopeptides, as previously disclosed in the following patents. U.S. Patent 4,062,831; U.S. Patent 4,097,470.

The polymeric conjugates of the present invention and the novel paclitaxel derivatives of formula (II) have excellent water solubility and very good biocompatibility and release paclitaxel or paclitaxel by cleavage of the oligopeptide hydro-moieties after incorporation into plasma or cells. derivative.

Biological activity

Microtubule assembly and disintegration assay

The two-cycle assembly disassembly method [Shelanski, M.L., Gaskin, F., and Cantor, C.R., Proc. Natl. Acad. Sci. USA, 70, 765-768 (1973)], and then prepared in MAB medium (0.1 M MES, 2.5 mM EGTA, 0.5 mM magnesium sulfate, 0.1 mM). EDTA, 0.1 mM DTT, pH 6.4) was stored under liquid nitrogen.

All experiments were performed on protein stored for less than four weeks.

Prior to the experiments, tubulin was stored at 4 ° C for 30 minutes.

The assembly was performed by Gaskin et al. method [Gaskin F.,

Cantor C.R., and Shelanski M.L., J. Molec. Bioi., 89, 737

-758 (1974)]. The cuvettes containing tubulin (1 mg / ml) and 1 mM GTP were set at 37 ° C and then maintained at 340 nm with a Perkin-Elmer 557 Double Wavelength Double Beam Spectrophotometer equipped with an automatic recorder and a thermostatically controlled sample chamber. turbidity measurements were performed.

After thirty minutes, 4 mM calcium chloride was added to the sample, and depolymerization was measured for 10 minutes as the turbidity decreased. Test compounds were added to the samples at regular intervals, in 15-minute increments, and a change in turbidity was observed. Data measured in this manner are expressed as the percentage of ripolymerization induced by the test compounds. The results are shown in Table 1.

In vitro drug sensitivity test

Exponentially growing B16-F10 murine melanoma cells (2 x 10 4 cells / ml) were placed in RPMI 1640 medium supplemented with thermally inactivated 10% fetal bovine serum and 2 mM glutamine in a 24-well culture plate (Costar). Immediately after insertion, test compounds were weighed in scalar concentrations. After incubating the samples for 72 hours, the cells were counted with a cell counter and the inhibition of cell growth was evaluated. Three cultures were used at each compound concentration tested. Dose response curves were used to calculate the antiproliferative activity of the test compounds expressed as IC5Q (dose required for 50% inhibition of cell growth in treated cultures as compared to untreated control). The results obtained are shown in Table 1.

Table 1

Compound Tubulin Composition% IC ₅₀ (μΜ) 0.5 μΜ 5 μΜ Example 1 0 15 19 Example 9 79 145 47 Example 3 46 86 23 Example 4 0 10 11 Example 6 0 10 51 Reference compound: paclitaxel 41 96 39

The copolymer paclitaxel prepared according to Example 6 was tested in vivo against B16 F10 murine melanoma compared to paclitaxel.

Mice

Female C57B16 mice were obtained from Charles River Italy.

The animals were 8 to 10 weeks old at the start of the experiments.

Active Ingredients

Because paclitaxel has a very limited water solubility, the compound is dissolved in a vehicle consisting of 50% by volume polyethoxylated castor oil (Cremophor EL) and 50% by volume ethanol, and the resulting solution is then diluted with 5% glucose solution to the desired concentrations. The solution was slightly cloudy and a noticeable precipitate formed after a short time.

The compound prepared according to Example 6 readily dissolved in 5% glucose solution and the resulting solution remained clear for a prolonged period (more than two hours). Final concentrations after dilution were based on the compound's paclitaxel content (up to 4%).

Tumor

Murine B16F10 melanoma cells were used in the experiments. Mice were injected subcutaneously with a 10 µl suspension of tumor cells in a volume of 0.2 ml. Tumor size was measured with a caliper, and tumor mass was calculated using the following equation:

the? + b

Administration of the active ingredients

Due to the poor solubility of paclitaxel and the toxicity of the vehicle, this drug was administered intraperitoneally.

Example 6 was administered by intravenous injection.

Both compounds are followed by tumor implantation

Treatment was performed on days 1, 5 and 9.

The data presented in Table 2 shows that the compound of the invention is significantly more active than paclitaxel.

The dose of the polymer conjugate is based on the paclitaxel content.

Table 2

Compound Dose (mg / kg) Tumor Inhibition (%) TOX control - - - paclitaxel 14, 6 53 0/10 22 38 0/10 33 92 1/7 Example 6 Compound 14, 6 77 0/9 22 79 0/10

TOX: number of animals killed due to toxicity / number of animals tested

Animals that had died or showed significant body weight loss and / or spleen and / or liver size reduction in the control animals were considered in the toxicity assessment.

From the above data it can be clearly seen that the polymer conjugates of the present invention have quite excellent antitumor activity. Due to their lower toxicity and increased water solubility compared to paclitaxel or paclitaxel derivatives, the compounds of the present invention are well suited for use as antitumor agents. Examples of tumors that can be treated with the polymeric conjugates of the invention include sarcomas, carcinoma, lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor, leukemias and adenocarcinoma.

The increased solubility and reduced toxicity of the polymer conjugates of the present invention means that the compounds are also suitable for intravenous injection or infusion.

The treatment dose depends on the age, weight and condition of the patient. The dosage of the active ingredient is generally in the range of 1 mg / kg to 1 g / kg body weight, preferably 4 to 800 mg / kg body weight. Typical formulations include the polymeric paclitaxel / polymeric paclitaxel derivative in an amount equivalent to 0.5, 1.5, 10, 20, 25 or 50 mg of the active paclitaxel / paclitaxel derivative.

The polymeric conjugates of the invention may be formulated into pharmaceutical compositions with pharmaceutically acceptable carriers or diluents. Any suitable carrier or diluent may be used. Solutions for intravenous injection or infusion may contain as vehicle or diluent, for example, sterile water, or preferably such solutions will be in the form of a sterile aqueous solution or isotonic saline.

The following Examples serve to illustrate the invention.

t *

- 19 Example 1

1-methacryloyl-1-amino-2-hydroxypropane, 1- [methacryloylglycyl (phenylalanyl) leucylglycyl] amino-2-hydroxypropane and 2-methacryloylglycyl (phenyl). copolymer of alanyl) -leucylglycyl] -paclitaxel

Copolymer of 1-methacryloylamino-2-hydroxypropane and N- [methylenecarbonyl (phenylalanyl) leucylglycyl (4-nitrophenoxy)] methacrylamide [1.4 g; the copolymer was prepared according to J. Kopecek et al., Makromol. Chem., 177, 2833 (1976)] with anhydrous dimethylformamide (15 mL) was added paclitaxel (100 mg) and dimethylaminopyridine (15 mg).

The yellow solution was stirred under anhydrous conditions for 8 hours at room temperature. Subsequently, 2-hydroxypropylamine (0.3 ml) was added dropwise to the reaction vessel and the reaction mixture was stirred for an additional 30 minutes.

To the reaction mixture was added glacial acetic acid (0.3 mL), the reaction mixture was quenched under reduced pressure and the residue was poured into acetone (200 mL).

After stirring for thirty minutes, the precipitate was filtered off and washed with acetone. 1.25 g of the title compound are obtained.

»·

The product had a paclitaxel content of 4.5% based on enzymatic hydrolysis and high performance liquid chromatography (HPLC).

Unreacted paclitaxel was recovered from the acetone solution.

Example 2

2 '- [N-Trityl (phenylalanyl) leucyl-glycyl] paclitaxel

To a solution of paclitaxel (170 mg) in acetonitrile (16 mL) was added dimethylaminopyridine (24 mg) and N-trityl (phenylalanyl) leucylglycine (4-nitrophenyl) ester (150 mg). .

The yellow solution was stirred at room temperature for 20 hours and then evaporated to dryness in vacuo.

The residue was chromatographed on silica gel with ethyl acetate / hexane (35:25, v / v). As a result, the title compound was obtained in an amount of 380 mg.

1 H-NMR (400 MHz, CDCl ₃ ) δ ppm:

0 82 (3H , d, J = 6.4 Hz, δ-Leu) 0 85 (3H , d, J = 6.7 Hz, δ-Leu) 1 15 (3H f S, 16) 1 26 (3H t S r 17) 1 2-1 , 6 (3H, m, β + β + γ-Leu).

J «k

Example 3

2 '- [(phenylalanyl) leucyl-glycyl] paclitaxel

The product obtained in the previous example, 2 '- [N-trityl (phenylalanyl) leucylglycyl] paclitaxel (250 mg) was dissolved in a mixture of glacial acetic acid (22 ml) and water (6 ml) and and stirred for one hour at room temperature.

After evaporation of the solvents in vacuo, the mixture was evaporated to dryness and the residue was stirred in diethyl ether / hexane (1: 1) for 30 minutes. After filtration, 160 mg of the title compound is obtained.

FAB-MS: m / z 1171, [M + H] ⁺ ; 1112, M-CH 3 COOH + 2H;

1051, 1024, 911, 603, 569, 509.

¹ H-NMR (400 MHz, CDCl 3) δ ppm:

0 88 (3H, d, J = 6.4 Hz, δ-Leu) 0 92 (3H, d, J = 6.4 Hz, δ'-Leu) 1 13 (3H, s, 16) 1 16 (3H, s, 17) 1 4-2 , 0 (4H, m, β + β '+ yLeu + 6β) 1 69 (3H, s, 19) 1 91 (3H, d, J = 1.2 Hz, 18) 2, 16 (1H, dd, J = 6.0 Hz , J = 13.8 Hz, 14 2, 23 (3H, s, CH 3 CO-IO) 2, 4-2 , 6 (3H, m, 6a + 14 + βΡίΐθ)

• · · ·

2.53 (3H, s, CH ₃ CO-4) 2, 90 (1H, dd, J = 4.1 Hz, J = 13 5 Hz, β'-Phe 3.49 (1H, dd, J = 4.0 Hz, J = 9, 1 Hz, aPhe) 3, 82 (1H, d, J = 7.3 Hz, 3) 3, 9-4 , 1 ( 2H, m, a + a'Gly ') 4.22 4.3 3 (2H, two d, J = 8, 7 Hz , CH ₂ -20) 4.27 (1H, m, a-Leu) 4.44 (1H, dd, J = 6.4 Hz, J = 10 8 Hz, 7) 4, 98 (1H, dd, J = 2.4 Hz, J = 9.7 Hz, 5) 5, 61 (1H, d, J = 3.2 Hz, 2 ') 5.70 (1H, d, J = 7.3 Hz, 2.) 6, 12 (1H, dd, J = 3.2 Hz, J = 9.4 Hz, 3 ') 6, 21 (1H, m, 13) 6, 28 (1H, s, 10)

6.8-8.2 (21Η, m, 4 Ph + NH-Leu)

7.87 (1H, d, J = 9.4 Hz, NH-4 ')

Example 4

1-Methacryloyl-1-amino-2-hydroxypropane, 1- (methacryloylglycyl) amino-2-hydroxypropane and 2 '- [methacryloylglycyl (phenylalanyl) leucylglycyl] - copolymer of paclitaxel

Copolymer of 1-methacryloylamino-2-hydroxypropane and N- [methylenecarbonyl- (4-nitrophenoxy)] - methacrylamide (1 g; copolymer known from

• ·

- prepared according to 23 procedures: P. Rejmanova et al., Makromol. Chem., 178, 2159-2168) with anhydrous dimethylformamide (10 mL) was added 2 '- [(phenylalanyl) leucylglycyl] paclitaxel (100 mg) and dimethylaminopyridine (10 mg).

The yellow solution was stirred under anhydrous conditions for 2 hours at room temperature. Subsequently, 2-hydroxypropylamine (0.15 mL) was added to the reaction vessel and the reaction mixture was stirred for an additional 30 minutes.

To the reaction mixture was added glacial acetic acid (0.2 mL), the reaction mixture was quenched under reduced pressure and the residue was poured into acetone (200 mL).

After stirring for one hour, the precipitate was filtered off and washed with acetone. 960 mg of the title compound were obtained.

The product had a paclitaxel content of 6% by enzymatic hydrolysis and HPLC.

Example 5

Copolymer of 1-methacryloylamino-2-hydroxypropane, 1- (methacryloylglycyl) amino-2-hydroxypropane and 2 (methacryloylglycyl) paclitaxel

Paclitaxel (100 mL) was added to a solution of a copolymer of 1-methacryloylamino-2-hydroxypropane and N- [methylene carbonyl (4-nitrophenoxy)] methacrylamide (1.6 g) in anhydrous dimethylformamide (16 mL). mg) and dimethylaminopyridine (20 mg) were added.

The yellow solution was stirred for 20 hours at room temperature. Subsequently, 2-hydroxypropylamine (0.2 mL) was added to the reaction vessel and the reaction mixture was stirred for an additional 30 minutes.

To the reaction mixture was added glacial acetic acid (0.3 mL), the reaction mixture was quenched under reduced pressure and the residue was poured into acetone (200 mL).

After stirring for one hour, the precipitate was filtered off and washed with acetone. 1440 mg of the title compound were obtained.

The product thus obtained had a paclitaxel content of 2.75 wt%.

• · »· ♦«

Example 6

1-Methacryloylamino-2-hydroxypropane, 1- [methacryloylglycyl (phenylalanyl) leucylglycyl] amino-2-hydroxypropane and 2 '- [methacryloylglycyl- ( copolymer of phenylalanyl) -leucylglycyl-Palanyl] -paclitaxel

Copolymer of 1-methacryloylamino-2-hydroxypropane and N- [methylene carbonyl (phenylalanyl) leucylglycyl (4-nitrophenoxy)] methacrylamide (620 mg) with anhydrous dimethylformamide ( 6 ml) of 2 '- (Palanyl) paclitaxel [62 mg; The compound was prepared according to the procedure described in N. F. Magri et al., J. Nat. 51: 298-306 (1988)] and dimethylaminopyridine (10 mg) were added.

The yellow solution was stirred under anhydrous conditions for 5 hours at room temperature. Subsequently, 2-hydroxypropylamine (0.1 ml) was added to the reaction vessel and the reaction mixture was stirred for an additional 30 minutes.

To the reaction mixture was added glacial acetic acid (0.15 mL), the reaction mixture was quenched under reduced pressure and the residue was poured into acetone (150 mL).

After stirring for one hour, the precipitate was filtered off and washed with acetone. 5.85 mg of the title compound were obtained.

t »4

The product thus prepared had a paclitaxel content of 4% by weight.

Example 7

2 ', 7-di (benzyloxy-carbonyl-p-alanyl) paclitaxel

To a solution of paclitaxel (200 mg) in acetonitrile (15 mL) was added N, N'-dicyclohexylcarbodiimide (400 mg), benzyloxycarbonyl-p-alanine (200 mg) and dimethylaminopyridine (60 mg). After stirring for 20 hours, the precipitate was filtered off and the filtrate was evaporated to dryness under reduced pressure.

The residue was chromatographed on silica gel with ethyl acetate / hexane (1: 1). As a result, the title compound was prepared in an amount of 300 mg.

FAB-MS: m / z 1264 [M + H] ⁺ , 1204, 1130, 1070.

Example 8

7- (Benzyloxy-carbonyl-p-alanyl) paclitaxel

To a solution of 2 ', 7-Di-benzyloxycarbonyl-p-alanyl-paclitaxel (171 mg) in methanol (60 mL) was added sodium bicarbonate (30 mg) and water (7 mL). The reaction mixture was stirred at room temperature for 3 hours.

I

The methanol was removed by evaporation and the aqueous residue was extracted with ethyl acetate.

The solvent was evaporated in vacuo and the extract was evaporated to dryness. As a result, 134 mg of the title compound were obtained.

Example 9

7- (β-alanyl) paclitaxel

To a solution of 7- (benzyloxycarbonyl-p-alanyl) paclitaxel (135 mg) in a mixture of methanol (20 mL) and formic acid (13 mL) was added 5% Pd / C (200 mg) catalyst. The reaction mixture was stirred at room temperature for 6 hours. The catalyst was filtered off, the filtrate was washed with methanol and the combined filtrate was evaporated to dryness by evaporation of the solvents under vacuum.

The residue thus obtained was dissolved in 8 ml of methanol and then precipitated with 150 ml of diethyl ether. As a result, the title compound was obtained in an amount of 85 mg.

FAB-MS: m / z 925, [M + H] ⁺ ; 947, [M + Na] ⁺ .

¹ H-NMR (300 MHz, CDCl ₃ ) δ ppm:

1, 14 (3H, and, CH3-16) 1.20 (3H, and, CH3-17) 1.79 (3H, And, CH3-19)

1.85 (3H, s, CH 3 -18) 2.17 (3H, s, CH 3 CO-10) 2.2-2 , 6 (6H, m, CH ₂ -14 + CH ₂ -6 + OCOCH ₂ CH ₂ NH ₂ 2.42 (3H, s, CH 3 CO-4) 3, 0-3 , 2 (2H, m, OCOCH ₂ CH ₂ nh ₂ ) 3.90 (1H, d, J = 6.8 Hz, 3) 4.18 4.31 (2H, two d, J = 8.2 Hz, CH ₂ -20) 4, 80 (1H, d, J = 3.2 Hz, 2 ') 4.91 (1H, d, J = 8.5 Hz, 5) 5, 62 (1H, dd, J = 10.2 Hz J = 7.0 Hz, 7) 5, 66 (1H, d, J = 6.8 Hz, 2.) 5.81 (1H, dd, J = 2.9 Hz, J = < 3, 1 Hz, 3 ') 6, 17 (1H, m, 13) 6, 19 (1H, s, 10) 7.3 to 8 , 2 (16H, m, NH-4 '+ 3 Ph)

Example 10

1-Methacryloylamino-2-hydroxypropane, 1- [methacryloylglycyl (phenylalanyl) leucylglycyl] amino-2-hydroxypropane and 7- [methacryloylglycyl (phenyl) -alanyl) -leucylglycyl-alanyl] -paclitaxel copolymer

Anhydrous dimer of a copolymer of 1-methacryloylamino-2-hydroxypropane and N- [methylene carbonyl (phenylalanyl) leucylglycyl (4-nitrophenoxy)] methacrylamide (1500 mg). ..5

To a solution of 29 formyl amide (13 mL) was added 7- (Palanyl) paclitaxel (135 mg) and dimethylaminopyridine (20 mg).

The yellow solution was stirred under anhydrous conditions for 5 hours at room temperature.

Subsequently, 2-hydroxypropylamine (0.2 mL) was added to the reaction vessel and the reaction mixture was stirred for an additional 30 minutes.

To the reaction mixture was added glacial acetic acid (0.3 mL), the reaction mixture was quenched under reduced pressure and the residue was poured into acetone (250 mL).

After stirring for one hour, the precipitate was filtered off and washed with acetone. 1520 mg of the title compound were obtained.

The product thus prepared had a paclitaxel content of 7.8% by weight.

Claims (10)

PATENT CLAIMS Polymer conjugate, characterized in that the conjugate comprises the following units: 90-99.9 mol% of the unit of formula (A); CH ₂ CH 3 -C-C 0 -NH-CH ₂ -CHOH-CH 3 (A) 0.1 to 5 mol% of a unit of formula (B) in which One of R 8 and R ₂ is a copolymer of formula (C), ch ₂ CH 3 -C-CONH-CH ₂ -CO-A- (C) while the other is hydrogen; and 0-9.9 mol% unit of formula (D), ♦ <··· ** • · ♦ ♦ « _{2 2} ch ₂ CH ₃ -c-CONH-CH ₂ -CO-A _x -NH-CH ₂ -CHOH-CH ₃ (D) where in the general formulas R is phenyl or tert-butoxy, R ₃ is hydrogen or acetyl, A and Ai, the same or different, represent a single bond, an amino acid residue, or one of the following peptide spacer groups (hydro moieties): PAla, Gly, Phe-Gly, Phe-Phe, Leu-Gly, Val-Ala, Phe-Ala , Leu-Phe, Leu-Ala, Phe-Leu-Gly, Phe-Phe-Leu, Leu-Leu-Gly, Phe-Tyr-Ala, Phe-Gly-Phe, Phe-Phe-Gly, Phe-Leu-Gly-Phe, Gly-Phe-Leu-Gly-Phe, Gly-PAla, Phe-Gly-3Ala, Phe-Phe-pAla, Leu-Gly ~ PAla, Val-Ala-pAla, Phe-Ala-pAla, Leu-Phe-pAla, Leu-Gly-pAla, Phe-Leu-Gly-Palais, Leu-Leu-Gly-Ala, Phe-Gly-Phe-βΑΙβ, Phe-Phe-Leu-3Ala, Phe-Tyr-Ala-Ala, Phe-Phe-Gly-Ala, You're Phe-Leu-Gly-Phe-pAla Gly-Phe-Leu-Gly-Phe-Ala. · * · Ί. ··. S ......: • · ♦ · ··· • · · «•« »« * «9 Polymer conjugate according to claim 1, characterized in that R is phenyl, R ₃ is acetyl, and A is Phe-Leu-Gly or Phe-Leu-Gly-pAla. Polymer conjugate according to claim 1 or 2, characterized in that the compound is 1-methacryloylamino-2-hydroxypropane, methacryloylglycyl (phenylalanyl) leucylglycyl] -3-. a copolymer of amino-2-hydroxypropane and 2 '- [methacryloylglycyl (phenylalanyl) leucylglycyl palanyl] paclitaxel. A process for the preparation of a polymeric conjugate according to claim 1, characterized in that a compound of the formula II in which One of ₂ and A ₃ is a chemical bond, while the other has the same meaning as A and A, R and R ₃ are as defined in claim 1 in an amount of 90 to 99.9 mol% of a unit of formula (A) I ch ₂ CH ₃ —C — CO — NH — CH ₂ —CHOH — CH ₃ (A) ··· «. ··. : ··· ♦ ··· · «· • · ♦ •• η« and from 10 to 0.1 mol% of a unit of formula (E) CH ₂ CH ₃ -C-CO-NH-CH ₂ -CO-A 1 -O- (1,4-phenylene) -NO ₂ (E) - in the formula Aj is a reaction with a stationary activated polymer as defined in claim 1 and the resulting polymer conjugate is then reacted with 2-hydroxypropylamine. Process according to claim 4, characterized in that the reaction is carried out in anhydrous polar solvent in the presence of an organic or inorganic base, at a temperature between 15 ° C and 40 ° C for 1-24 hours. 6. A compound of formula (II) as defined in claim 4 wherein: ₂ is the same as the di-, tri- or tetrapeptide bridge moiety as defined in claim 1. ··· «. ··. : ··· • ···> ·· * · r 7. A process for the preparation of compounds of formula II according to claim 6, characterized in that paclitaxel or a paclitaxel analogue is reacted with a protected amino acid or a protected peptide and a condensing reagent or activated esters and optionally in the presence of a catalyst. protecting groups are removed. Compounds of formula (II) according to claim 4, characterized in that A3 is a group PAa or the di-, tri- or tetrapeptide hydroxy moiety as defined in claim 1 for A. 9. A process for the preparation of compounds of formula II as defined in claim 8, wherein (i) the 2'-hydroxy group of paclitaxel is protected or blocked, followed by esterification of the 7-position hydroxy group and removal of the 2'- or (ii) paclitaxel is reacted with 2-3 equivalents of the N-protected desired amino acid, and the 2 ', 7-disubstituted taxol thus obtained is cleaved at the 2' position and subsequently removed at the 7-position amino acid. protecting group. •• ·· ·· ···· ···. · · * «· · Í í:: 10. A pharmaceutical composition, wherein the active ingredient is one of claims 1, 6 or The polymer conjugate of any one of claims 8 to 8 together with a pharmaceutically acceptable diluent or carrier.